NO331809B1 - Neurotrophic growth factor - Google Patents

Abstract

An isolated nucleic acid molecule is described which encodes a human neurotrophic growth factor called enovin, and which has the amino acid sequence illustrated in Figures 1, 21, 23 or 24, or which encodes a functional equivalent, a derivative or a bioprocessor of said growth factor. The preferred growth factor comprises the amino acid sequence from positions 27 to 139 of the sequence illustrated in Figure 1, or a functional equivalent, a derivative or a bioprocessor thereof. The nucleic acid molecule encoding enovin can be used to transform a host cell, tissue or organism by including it in a suitable vector. The host cell, tissue or organism and vector also form part of the invention.

Description

NEUROTROPHIC GROWTH FACTOR

The present invention relates to a neurotrophic factor, in particular the cloning and expression of a new member of the GDNF family of neurotrophic factors, designated herein as "enovin" (EVN).

Introduction

Neurotrophic factors are involved in neuronal differentiation, development and maintenance. These proteins can prevent degeneration and promote survival of different types of neuronal cells and are thus potential therapeutic agents for neurodegenerative diseases. Glial cell line-derived neurotrophic factor (GDNF) was the first member of a growing subfamily of neurotrophic factors that are structurally distinct from the neurotrophins. GDNF is a member of the transforming growth factor β superfamily (TGF-β) of growth factors, characterized by the specific pattern of seven highly conserved cysteine residues within the amino acid sequence (Kingsley, 1994). GDNF was originally purified using an assay based on its ability to maintain the survival and function of embryonic ventral midbrain dopaminergic neurons in vitro (Lin et al., 1993). Other neuronal cell types in the central (CNS) or peripheral nervous system (PNS) are also responsive to the survival effects of GDNF (Henderson et al., 1994, Buj-Bello et al., 1995, Mount et al., 1995, Oppenheim et al. , 1995). GDNF is produced by cells in an inactive proform, which is cleaved specifically at an RXXR-furin recognition site to produce active (fully developed) GDNF (Lin et al., 1993). Exogenous administration of GDNF has potent neuroprotective effects in animal models of Parkinson's disease, a common neurodegenerative disorder characterized by the loss of up to 70% of dopaminergic cells in the substantia nigra of the brain (Beck et al., 1995, Tomac et al., 1995, Gash et al., 1996, Choi-Lundberg et al., 1997, Bilang-Bleuel et al., 1997).

Recently, additional neurotrophic factors of the GDNF family have been discovered. Neurturin (NTN) was purified from conditioned medium of Chinese hamster ovary (CHO) cells using an assay based on its ability to promote the survival of sympathetic neurons in culture (Kotzbauer et al., 1996). The fully developed NTN protein is 57% similar to the fully developed GDNF. Persephin (PSP) was discovered by cloning using degenerate primer PCR with genomic DNA as template. The fully developed PSP, like the fully developed GDNF, promotes the survival of ventral midbrain dopaminergic neurons and of motor neurons in cultures (Milbrandt et al., 1998). The similarity between the fully developed PSP protein with fully developed GDNF and NTN is w 50%. Artemin (ARTN) was discovered by DNA database search and is a survival factor of sensory and sympathetic neurons in cultures (Baloh et al, 1998b).

GDNF, NTN, PSP and ARTN require a heterodimeric receptor complex to carry out downstream intracellular signal transduction. GDNF binds to the GDNF family receptor alpha 1 (GFRa-1; "GFRa Nomenclature Committee", 1997) subunit,

a glycosyl-phosphatidyl-inositol (glycosyl-Ptdlns)-anchored membrane protein (Jing et al., 1996, Treanor et al., 1996, Sanicola et al., 1997). The GDNF/GFRα-1 complex then binds to and activates the cRET proto-oncogene, a membrane-bound tyrosine kinase (Durbec et al., 1996, Trupp et al., 1996), resulting in phosphorylation of tyrosine residues in cRET and subsequent activation of downstream signal transduction pathways (Worby et al., 1996). Several other members of the GFRα family of ligand-binding receptors have been characterized (Baloh et al., 1997, Sanicola et al., 1997, Klein et al., 1997, Buj-Bello et al., 1997, Suvanto et al., 1997). GFRα-2 and GFRα-3 (Jing et al., 1997, Masure et al., 1998, Woby et al., 1998, Naveilham et al., 1998, Baloh et al., 1998a) have been identified by a number of different groups. GFRa-1 and GFRa-2 are expressed to a large extent in almost all tissues, and the expression can be regulated

are learned developmentally (Sanicola et al., 1997, Widenfalk et al., 1997).

GFRα-3 is not expressed in the developing or adult central nervous system, but is highly expressed in several developing and adult sensory and sympathetic ganglia in the peripheral nervous system (Widenfalk et al., 1998, Naveilhan et al., 1998, Baloh et al., 1998a). A fourth family member, GFRα-4, was cloned from chicken cDNA (Thompson et al., 1998). GFRα-1 is the preferred receptor for GDNF, whereas GFRα-2 preferentially binds NTN (Jing et al., 1996, Treanor et al., 1996, Klein et al., 1997). Chicken GFRα-4 forms a functional receptor complex for PSP in combination with cRET (Enokido et al., 1998). Cells expressing both GFRα-3 and cRET were found to be unresponsive to either GDNF, NTN or PSP (Worby et al., 1998, Baloh et al., 1998a). More recently, ART has been shown to signal via cRET using GFRα-3 as the preferred ligand-binding receptor (Baloh et al., 1998b). Cross-communication between the neurotrophic factors and the GFRα receptors is possible in vitro, as GDNF can bind to GFRα-2 or GFRα-3 in the presence of cRET (Sanicola et al., 1997, Trupp et al., 1998), and NTN can bind to GFRα-1 with low affinity (Klein et al., 1997). Briefly, GDNF, NTN, PSP and ART are part of a neurotrophic signaling system whereby different ligand-binding subunits (GFRα-1 to -4) can interact with the same tyrosine kinase subunit (cRET). The physiological relevance of these in vitro findings was recently demonstrated in gene knockout studies (reviewed by Rosenthal, 1999), which clearly show that GDNF interacts with GFRα-1 in vivo, whereas NTN is the preferred ligand for GFRα- 2.

The present inventors have identified, cloned, expressed, chromosomally located and characterized Enovin (EVN), the fourth member of the GDNF family. Knowledge of the fully developed EVN protein has been expanded with the discovery of various functional and non-functional mRNA splice variants. Furthermore, we present expression data, binding data of EVN to GFRα-3 and in vitro effects of EVN on neurite outgrowth and protection against taxol-induced neurotoxicity in staurosporine-differentiated SH-SY5Y human neuroblastoma cell cultures.

Summary of the invention

The present application provides a nucleic acid molecule encoding a new human neurotrophic growth factor, "enovin", an expression vector comprising said nucleic acid molecule, a host cell transformed with said vector, a neurotrophic growth factor encoded by said nucleic acid molecule, isolated enovin, and pharmaceutical compositions containing the nucleic acid or the enovin protein or the agonists or antagonists thereof.

Detailed description of the invention

According to a first aspect of the present invention, a nucleic acid molecule is provided which encodes a human neurotrophic growth factor, designated herein as enovin, which has the amino acid sequence illustrated in Figure 21, or which encodes a functional equivalent, a derivative or a bioprecursor of said growth factor. Preferably said nucleic acid molecule is DNA and even more preferably a cDNA molecule.

Preferably, the nucleic acid according to the invention comprises the sequence from positions 81 to 419 of the sequence illustrated in Figure 1 and more preferably from positions 81 to 422 and even more preferably the complete sequence illustrated in Figure 1.

The nucleic acid molecule from position 81 to 419 is assumed to encode the sequence of the fully developed enovin protein after the production of the pro-form of the protein at the RXXR process site which is present in the stable pro-form of said enovin protein.

Advantageously, the nucleic acid molecule according to the invention can be used to express the human neurotrophic growth factor according to the invention, in a host cell or the like using a suitable expression vector.

An expression vector according to the invention comprises vectors which are capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, which are capable of causing expression of such DNA fragments.

Regulatory elements required for expression include promoter sequences for RNA polymerase binding and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector may comprise a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector may comprise a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the initiation codon AUG, and a termination codon for ribosome release. Such vectors can be obtained commercially or assembled from sequences described by methods well known in the art.

Thus, an expression vector refers to a recombinant DNA or RNA composition, such as a plasmid, a phage, recombinant virus or another vector which, when introduced into a suitable host cell, results in expression of the DNA or RNA fragments. Suitable expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those that integrate into the host cell genome.

The nucleic acid molecules according to the invention can be inserted into the vectors described in an antisense orientation to ensure the production of antisense RNA. Antisense RNA or other antisense nucleic acids can be produced synthetically.

A further aspect of the invention comprises the host cell transformed, transfected or infected with the expression vector according to the invention, which cell preferably comprises a eukaryotic cell and more preferably a mammalian cell.

Incorporation of the cloned DNA into a suitable expression vector for subsequent transformation of said cell and subsequent selection of the transformed cells is well known to those skilled in the art, as described in Sambrook et al

(1989) "Molecular Cloning", A Laboratory manual, Cold Spring Harbor Laboratory Press.

A further aspect of the present invention comprises a nucleic acid molecule which has at least 15 nucleotides of the nucleic acid molecule according to the invention and preferably from 15 to 50 nucleotides.

These sequences can advantageously be used as probes or primers to initiate replication or the like. Such nucleic acid molecules can be prepared according to techniques well known in the art, such as by recombinant or synthetic means. They can also be used in diagnostic equipment ("kits") or devices or the like to detect the presence of a nucleic acid according to the invention. These tests generally involve contacting the probe with a sample under hybridization conditions and detecting the presence of any duplex formation between the probe and any nucleic acid in the sample.

According to the present invention, these probes can be anchored to a solid carrier. Preferably, they are present in a regular line position so that multiple probes can simultaneously hybridize to a single biological probe. The probes can be localized into the array or synthesized in situ on the array. (See Lockhart et al., Nature Biotechnology, vol. 14, December 1996 "Expression Monitoring by Hybridization in High Density Oligonucleotide Arrays". A single array may contain more than 100, 500 or even 1000 different probes in distinct locations.

The nucleic acid molecules according to the invention can also be produced using such recombinant or synthetic means, such as for example using PCR cloning mechanisms which generally involve creating a pair of primers, which can be from approximately 10 to 50 nucleotides to a region of the gene which it is desired to clone, by contacting the primers with mRNA, cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions that induce amplification of the desired region, isolating the amplified region or fragment, and recovering it amplified DNA. In general, such techniques as are defined herein are well known in the art, as described in Sambrook et al (Molecular Cloning: a Laboratory Manual, 1989).

The nucleic acids or oligonucleotides according to the invention may carry a revealing label. Suitable labels include radioisotopes such as 32 P or<35>S, enzyme labels or other protein labels such as biotin or fluorescent markers. Such labels can be added to the nucleic acids or oligonucleotides according to the invention and can be detected using techniques known per se.

Advantageously, human allelic variants or polymorphisms of the DNA molecule according to the invention can be identified by, for example, probing cDNA or genomic collections from many different individuals, for example from different populations. Furthermore, nucleic acids and probes according to the invention can be used to sequence genomic DNA from patients using techniques well known in the art, such as Sanger's dideoxy chain termination method, which can advantageously ascertain any predisposition in a patient associated with certain disorders with a growth factor according to the invention.

Furthermore, the present invention provides a transgenic cell, a tissue or an organism comprising a transgene capable of expressing the human neurotrophic factor enovin according to the invention.

The term "transgene capable of expression" as used herein means any suitable nucleic acid sequence leading to the expression of a neurotrophic factor having the same function and/or activity of the neurotrophic factor of the invention. The transgene can comprise, for example, genomic nucleic acid isolated from human cells or synthetic nucleic acid comprising cDNA, integrated into the chromosome or in an extrachromosomal state.

Preferably, the transgene comprises a vector according to the invention, which vector comprises a nucleic acid molecule encoding said neurotrophic factor, or a functional fragment of said nucleic acid molecule. A "functional fragment" of said nucleic acid should be understood to mean a fragment of the gene or cDNA encoding said neurotrophic factor or a functional equivalent thereof, which fragment is capable of being expressed to produce a functional neurotrophic growth factor according to the invention. Thus, for example, fragments of the neurotrophic growth factor according to the invention that correspond to the specific amino acid residues that interact with the corresponding receptor also form part of the present invention, which fragments can serve to function as agonists that activate the corresponding receptor of the growth factor according to the invention to trigger its growth-promoting and survival-preserving effects on cells. This aspect of the invention also includes differentially spliced isoforms and the transcription connections of the nucleic acids according to the invention.

In accordance with the present invention, a defined nucleic acid includes not only the identical nucleic acid but also any minor base variation including especially substitutions in bases that result in a synonymous codon (a different codon specifying the same amino acid residue) due to the degenerative code in conservative amino acid substitutions. The term "nucleic acid molecule" also includes the complementary sequence to any single given stranded sequence in terms of base variations.

According to a further aspect, the invention provides an isolated human neurotrophic growth factor encoded by a nucleic acid molecule as defined herein. Preferably, the growth factor comprises an amino acid sequence from position 27 to 139 of the amino acid sequence in Figure 1 or a functional equivalent, a derivative or a bioprecursor thereof.

A "functional equivalent" as defined herein should be understood to mean a growth factor that exerts all the growth properties and functionality associated with the growth factor enovin. A "derivative" of enovin as defined herein is intended to include a polypeptide in which certain amino acids have been changed or removed or replaced with other amino acids, which polypeptide retains the biological activity of enovin, and/or which polypeptide can react with antibodies raised using enovin according to the invention as the challenging antigen.

Included within the scope of the present invention are hybrid forms and modified forms of enovin, including fusion proteins and fragments. The hybrid forms and the modified forms include, for example, when certain amino acids have been subjected to some modification or replacement, such as for example by point mutation, which modification nevertheless results in a protein which retains the biological activity of the enovine according to the invention. Specific nucleic acid sequences can be altered by those skilled in the art to produce a growth factor that exerts the same, or substantially the same, properties as enovin.

As is well known in the art, many proteins are produced in vivo with a (pre)signal sequence at the N-terminus of the protein. Furthermore, such proteins may comprise an additional pro-sequence which represents a stable precursor to the fully developed protein. Such pre- and pro-sequences are not generally required for biological activity. The enovin molecule according to the invention includes not only the entire length sequence illustrated in Figure 21, but from position 27 to 139, which follows the RXXR proteolytic processing site present in growth factors of this type, and which is believed to represent the fully developed sequence of enovin.

A defined protein, a polypeptide or an amino acid sequence according to the invention includes not only the identical amino acid sequence, but isomers thereof in addition to minor amino acid variations from the natural amino acid sequence including conservative amino acid substitutions (a substitution of an amino acid that is related in its side chains). Also included are amino acid sequences that vary from the natural amino acid, but result in a polypeptide that is immunologically identical or similar to the polypeptide encoded by the naturally occurring sequence.

Proteins or polypeptides according to the invention further comprise variants of such sequences, including naturally occurring allelic variants which are mainly homologous to said proteins or polypeptides. In this connection, homology is mainly considered as a sequence having at least 70%, and preferably 80%, 90% or 95%, amino acid homology with the proteins or polypeptides encoded by the nucleic acid molecules according to the invention.

Neurotrophic growth factors expressed by the host cells according to the invention are also included within the present invention.

Due to the sequence similarity of the growth factor described herein with previously identified growth factors of the GDNF family, it is believed that enovin is also capable of promoting cell survival and growth and may be used to treat disorders resulting from defects in function or expression of said neurotrophic factor.

The nucleic acid molecules or the neurotrophic factor according to the invention can therefore be advantageously used to treat or prevent neural disorders in an individual by administering to said individual an amount of a nucleic acid molecule or a growth factor according to the invention in sufficient concentration to reduce the symptoms of said disturbance. Thus, the nucleic acid molecules according to the invention can be used to promote the maintenance and survival of neuronal cells and to treat neuronal disorders or neurodegenerative conditions including Parkinson's disease, Alzheimer's disease, peripheral neuropathy, amyotrophic lateral sclerosis, peripheral and central nerve trauma or damage from exposure to neurotoxins.

It has advantageously been observed that the neurotrophic growth factor according to the invention has a neurotrophic or neuroprotective effect on neuronal cells or cell populations, especially those neuronal cells or cell populations that have been induced to undergo apoptosis. Accordingly, the nucleic acid or the enovin growth factor itself according to the invention can also be used to treat neurodegenerative disorders such as stroke, Huntingdon's disease, peripheral neuropathy, acute brain injury, nervous system tumors, multiple sclerosis, amyotrophic lateral sclerosis, peripheral nerve trauma, damage from exposure to neurotoxins, multiple endocrine neoplasia, familial Hirschsprung's disease, prion-related diseases, Creutzfeld-Jacob disease, by administering to a patient in need thereof, an amount of said nucleic acid or enovin in a concentration sufficient to reduce or prevent the symptoms of the neural disorders described herein.

In addition, and as described in more detail in the examples below, enovin has been shown to accelerate the reacquisition of induced sensory deficits, identifying enovin as a candidate for treating or ameliorating pain syndromes with a peripheral or central neurogenic component, rheumatic/inflammatory diseases as well as conductance disorders, when administered to a patient in need thereof in sufficient concentration to reduce or prevent the symptoms of these disorders.

An alternative method for treating the nerve disorders described above comprises implanting in an individual cells expressing a human neurotrophic growth factor according to the invention, such as the transgenic cell described herein.

The nucleic acid molecules and the neurotrophic growth factor according to the invention can also be incorporated into a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent or excipient.

Antibodies to the neurotrophic factor according to the present invention can advantageously be produced by techniques known in the art. For example, polyclonal antibodies can be prepared by inoculating a host animal such as a mouse with the growth factor or an epitope thereof and obtaining immune serum. Monoclonal antibodies can be prepared according to known techniques such as described by Kohler R. and Milstein C, Nature (1975) 256, 495-497.

Antibodies according to the invention can advantageously be used in a method for detecting the presence of a growth factor according to the invention, which method comprises reacting the antibody with a sample and identifying any protein bound to said antibody. An equipment ("kit") is also provided to carry out said method comprising an antibody according to the invention and means for reacting the antibody with said probe.

The present invention also provides an equipment or device for detecting the presence of a neurotrophic growth factor according to the invention in a sample, comprising an antibody as described above and means for reacting said antibody and said probe.

Proteins that interact with the neurotrophic factor according to the invention, such as for example its corresponding cellular receptor, can be identified by examining protein-protein interactions using the two-hybrid vector system that is well known to molecular biologists (Fields&Song, Nature 340:245 1989). This technique is based on functional reconstitution in vivo of a transcription factor that activates a reporter gene. More particularly, the technique comprises providing a suitable host cell with a DNA assembly comprising a reporter gene under the control of a promoter regulated or a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA sequence which encodes a first fusion of a fragment or all of a nucleic acid sequence according to the invention and either said DNA-binding domain or said activating domain of the transcription factor, express in the host at least one other hybrid DNA sequence, such as a collection or the like, which encodes putative binding proteins to be investigated, together with the DNA binding or activating domain of the transcription factor not incorporated into the first fusion; detecting any binding of the proteins to be examined with a protein according to the invention by examining the presence of any reporter gene product in the host cell; possibly isolating other hybrid DNA sequences that encode the binding protein.

An example of such a technique uses the GAL4 protein in yeast. GAL4 is a transcriptional activator of galactose metabolism in yeast and has a separate domain for binding to activators upstream of galactose metabolizing genes as well as a protein binding domain. Nucleotide vectors can be constructed, one of which comprises the nucleotide residues encoding the DNA-binding domain of GAL4. These binding domain residues can be fused to a known protein coding sequence, such as, for example, the nucleic acids according to the invention. The second vector comprises the residues encoding the protein-binding domain of GAL4. These residues are fused to residues encoding a test protein, preferably from the signal transduction reaction pathway of the host in question. Any interaction between the neurotrophic factor encoded by the nucleic acid according to the invention and the protein to be tested leads to transcriptional activation of a reporter molecule in a GAL-4 transcription-deficient yeast cell into which the vectors have been transformed. Preferably, a reporter molecule such as β-galactosidase is activated upon restoration of transcription of the yeast galactose metabolism genes.

The receptor for enovin has been identified by the present inventors as GFRα3. Assays can therefore be performed to identify agonist or antagonist compounds of enovin. This assay can also be used in conjunction with other neurotrophic growth factors and their corresponding receptors. Identified compounds can be used to treat or prevent disorders such as Parkinson's disease, Alzheimer's disease, neuronal disorders associated with expanded polyglutamine sequences, such as Huntingdon's disease, peripheral neuropathy, acute brain injury, nervous system tumors, multiple sclerosis, amyotrophic lateral sclerosis, peripheral nerve trauma or injury by exposure to neurotoxins, multiple endocrine neoplasia and familial Hirschsprung's disease, prion-related diseases, Creutzfeld-Jacob disease, stroke, pain syndromes with a predominantly peripheral or central neurogenic component, rheumatic/inflammatory diseases as well as conductance disorders, by administering to an individual an amount of said agonist or antagonist in sufficient concentration to prevent or treat said neural disorders. Such compounds can also be incorporated into pharmaceutical compositions together with a pharmaceutically acceptable carrier, diluent or excipient.

Agonists or antagonists of a growth factor (such as, for example, enovin) can be identified in one embodiment by contacting a tissue or organism expressing an appropriate receptor and cRET with a candidate compound, in the presence of the growth factor, and comparing the levels of RET -activation in said cell, tissue or organism with a control that has not been in contact with said candidate compound.

An alternative embodiment of the invention comprises a method for identifying agonists or antagonists of a neurotrophic growth factor, wherein said method comprises bringing a cell tissue or an organism expressing a suitable receptor of said growth factor and cRET into contact with a candidate compound in the presence of said growth factor, measuring the level of activation of a signaling kinase in the signal transduction reaction pathway, of which said appropriate receptor is a component, after the addition of an antibody specific for said signaling kinase conjugated to a reporter molecule, compared to a cell tissue or organism that has not been in contact with said connection.

A further aspect of the invention comprises the use of a compound identified as an antagonist according to the invention in the manufacture of a medicament for treating gastrointestinal disorders or conditions mediated by increased peristaltic intestinal movement. The compounds identified in the assays according to the present invention can advantageously be used to enhance gastrointestinal motility, and they can therefore be useful for treating conditions related to an inhibited or impaired gastrointestinal passage.

Accordingly, such compounds may be useful in treating warm-blooded animals, including humans, suffering from conditions related to an inhibited or impaired gastric emptying or, more generally, suffering from conditions related to an inhibited or impaired gastrointestinal passage. Accordingly, a treatment method is provided for curing patients from conditions such as, for example, gastroesophageal reflux, dyspepsia, gastroparesis, post-operative ileus and intestinal pseudo-obstruction.

Dyspepsia is a deterioration of digestive function, which can occur as a symptom of a primary gastrointestinal dysfunction, especially a gastrointestinal dysfunction related to an increased muscle tone or as a complication due to other disorders such as appendicitis, cholecystitis or malnutrition. Dyspeptic symptoms are, for example, lack of appetite, feeling full, premature satiety, nausea, vomiting and bloating.

Gastroparesis can occur with an abnormality in the stomach or as a complication of diseases such as diabetes, progressive systemic sclerosis, anorexia, nervosa and myotonic dystrophy.

Post-operative ileus is an obstruction or a kinetic deterioration in the viscera due to a disruption in muscle tone after an operative procedure.

Intestinal pseudo-obstruction is a condition characterized by constipation, colicky pain and vomiting, but without signs of physical obstruction.

The compounds according to the present invention can thus be used either to remove the actual cause of the condition or to alleviate the symptoms of the condition.

Moreover, some of the compounds, being stimulators of colonic kinetic activity, may be useful in normalizing or improving intestinal passage in individuals suffering from symptoms related to disturbed motility, e.g. reduced peristalsis in the small and large intestine, individually or in combination with delayed gastric emptying.

In light of the colon's kinetic utilization of the compounds according to the present invention, a method is provided for treating warm-blooded animals, including humans, suffering from motility disorders in the intestinal system, such as, for example, constipation, pseudo-obstruction, intestinal atonia, post-operative intestinal atonia , irritable bowel syndrome (IBS), and drug-induced delayed transit.

Compounds identified as antagonists according to the assays of the present invention may also be of potential use in the treatment or prophylaxis of gastrointestinal conditions resulting from increased peristaltic movements in the intestines such as diarrhea (including secretory diarrhea, bacterially induced diarrhea, colic diarrhea, traveler's diarrhea and psychogenic diarrhoea), Crohn's disease, spastic colon, irritable bowel syndrome (IBS), diarrhoea-predominant irritable bowel gastrointestinal hypersensitivity.

In light of the utilization of the compounds of the invention, it follows that the present invention also provides a method of treating warm-blooded animals, including humans, suffering from gastrointestinal conditions such as irritable bowel syndrome (IBS), particularly the diarrheal aspects of IBS. Accordingly, a method of treatment is provided for ameliorating patients suffering from conditions such as irritable bowel syndrome

(IBS), diarrhea-predominant irritable bowel syndrome, intestinal hypersensitivity and reduction of pain associated with gastrointestinal hypersensitivity.

The present compounds may also be of potential use in other gastrointestinal disorders, such as those associated with upper intestinal motility, and as antiemetics to treat emesis and cytotoxic drug- and radiation-induced emesis.

Inflammatory bowel diseases include, for example, ulcerative colitis, Crohn's disease and the like.

A further aspect of the invention also comprises a method of treating a disorder mediated by expression of enovin according to the invention, by administering to a patient an amount of an antisense molecule or an antagonist thereof according to the invention in a concentration sufficient to alleviate or reduce the symptoms of said disorder.

Disorders mediated by inactivation or inhibitory expression of enovin may also be advantageously treated by administering to an individual an amount of a compound identified as an agonist of enovin in a concentration sufficient to reduce or prevent the symptoms of the disorder.

In a further aspect, the invention provides a method of preparing a pharmaceutical formulation for treating diseases associated with the human neurotrophic growth factor enovin, said method comprising selecting a candidate compound identified as an agonist or antagonist of enovin according to the invention, to preparing bulk quantities of said compound and formulating the compound prepared in a pharmaceutically acceptable carrier. As shown in more detail from the examples below, enovin has been successful in reducing taxol-induced sensory deficits. Enovin may therefore play a possible role in pain syndromes with a mainly peripheral and central neurogenic component, rheumatic diseases as well as conductance disorders and may play a modulatory role in sensory processes after transdermal, topical, local central (such as epidural, intrathecal, ICV, intraplexus, intraneuronal ) peroral, rectal and systemic use. Therefore, similarly as described herein for other conditions mediated by enovin, these conditions can be alleviated or even prevented by administering either an antisense molecule, a nucleic acid, an enovin protein, a pharmaceutical composition, or a compound identified as an agonist or an antagonist, as needed, according to the invention, in sufficient concentrations to alleviate or prevent the symptoms of said disorder(s).

The therapeutic or pharmaceutical compositions of the present invention may be administered by any suitable means known in the art, including, for example, intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral administration, or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of a slow release formulation. To treat tissue in the central nervous system, administration may be by injection or infusion into the cerebrospinal fluid (CSF).

Enovin may also be bound or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. For example, it may be linked to any substance known in the art to promote penetration or transport through the blood-brain barrier such as an antibody to the transferrin receptor, and administered by intravenous injection.

Enovine, the antisense molecules or indeed the compounds identified as agonists or antagonists of enovine according to the invention, can be used in the form of a pharmaceutical composition, which can be prepared according to procedures well known in the art. Preferred compositions comprise a pharmaceutically acceptable vehicle or diluent or excipient, such as, for example, a physiological saline solution. Other pharmaceutically acceptable carriers, including other non-toxic salts, sterile water or the like, may also be used. A suitable buffer may also be present, which enables the compositions to be lyophilized and stored in a sterile condition prior to reconstitution by the addition of sterile water for subsequent administration. Incorporation of enovin into a solid or semi-solid biologically compatible matrix can be performed, which can be implanted into tissue in need of treatment.

The carrier may also contain other pharmaceutically acceptable excipients to modify other conditions such as pH, osmolarity, viscosity, sterility, lipophilicity, solubility or the like. Pharmaceutically acceptable excipients which enable sustained or delayed release after administration may also be included.

The enovin protein or nucleic acid molecules or compounds of the invention can be administered orally. In this embodiment, they can be encapsulated and combined with suitable carriers in solid dosage forms, which should be well known to those skilled in the art.

As should be well known to those skilled in the art, the specific dosage regimen may be calculated according to the patient's body surface area or volume of body space to be occupied, depending on the particular route of administration to be employed. However, the amount of composition actually administered will be determined by a medical practitioner, based on the circumstances of the disorder to be treated, such as the severity of the symptoms, the composition to be administered, the individual patient's age, weight and response, and the chosen route of administration .

The present invention can be better understood by the following examples, which are purely exemplary, and by reference to the accompanying drawings, in which: Figure 1 is a partial cDNA sequence of a neurotrophic factor according to the invention designated as enovin. The consensus sequence was obtained by PCR amplification with the primers PNHsp3 and PNHapl on different cDNAs and on genomic DNA followed by cloning and sequence analysis and comparison of the obtained sequences. The predicted one-letter coded amino acid sequence is shown above the DNA sequence. The nucleotide residue number is shown on the right side of the DNA sequence, while the amino acid residue number is shown on the right side of the translated protein sequence. The putative RXXR cleavage site for the prodomains is indicated in bold and underlined. The presumed beginning of the fully developed protein is indicated by an arrow. The seven conserved cysteine residues characteristic of all members of the TGF-β family are indicated in bold. A potential N-glycosylation site is doubly underlined, Figure 2 is an array of the predicted fully unfolded protein sequences of human GDNF, NTN, PSP and EVN. The sequences were aligned using the ClustalW alignment program. Conserved amino acid residues between all three proteins are included in the black areas. Conserved residues between two or three of the sequences are shaded in grey. The 7 conserved cysteine residues characteristic of members of the TGF-β family are indicated by asterisks above the sequence. The amino acid residues are numbered to the right. The dashes indicate spaces introduced in the sequence to optimize the alignment, Figure 3 is a partial cDNA sequence of enovin. The consensus sequence was obtained by PCR amplification (primary PCR with primers PNHsp1 and PNHapl and stranded PCR with primers PNHsp2 and PNHap2) on different cDNAs followed by cloning and sequence analysis and comparison of the obtained sequences. The translated one-letter coded amino acid sequence of nucleotides 30 to 284 (reading frame A) is shown above the sequence and numbered to the right (A1 to A85). This reading frame contains a putative ATG translation start codon. The translated one-letter encoded amino acid sequence of nucleotides 334 to 810 (reading frame B) is shown above the sequence and numbered to the right (B1 to B159). This reading frame contains the region of homology to GDNF, NTN and PSP. The nucleotide residue number is shown to the right of the DNA sequence. The putative RXXR cleavage site for the prodomains is indicated in bold and underlined. The presumed beginning of the fully developed protein is indicated by an arrow. The seven conserved cysteine residues characteristic of all members of the TGF-β family are indicated in bold. A potential N-glycosylation site is double underlined,

Figure 4 is an illustration of the chromosomal localization of human Enovin. (A) Diagram of FISH mapping results for Enovin. Each dot represents the double FISH signals detected on the human chromosome 1, region p31.3-p32.

(B) Example of FISH mapping of Enovin. The left field shows the FISH signals on chromosome 1. The right field shows

the same mitotic Figure stained with 4',6-diamidino-2-phenylindole to identify chromosome 1,

Figure 5 is an illustration of the expression of Enovin in various human tissues. (A), (B), (C) Northern blot analysis of tissue expression of Enovin. The expression of Enovin mRNA in various human tissues was assessed using a probe corresponding to part of the coding region of Enovin (including the region coding for the full-length Enovin protein) to analyze "blots" of human poly(A)-rich RNA . (A) "Multiple Tissue Northern (MTN) blot"; (B) “MTN-blot II”, Fetal MTN-blot II. Panel (D) shows an autoradiograph of the human RNA master blot probed with the same Enovin cDNA fragment. Panel (E) shows the localization of mRNEn samples from human tissue on the RNA master blot from (D),

Figure 6 is a graphical illustration of the overall survival of SH-SY5Y cells after 72 hours of treatment with 10 -6 M taxol and the effect of increasing doses of enovin on this survival, normalized to the solvent condition. SH-SY5Y cells are differentiated for 5 days with 25nM staurosporine before application of taxol. The data are from two independent experiments performed six times. Means and standard deviations are shown, Figure 7 is a graphical representation of the effect of increasing concentrations of enovin for 48 hours on a neurite outgrowth of staurosporine-differentiated SH-SY5Y cells, normalized to the solvent condition. The SH-SY5Y cells are differentiated for 5 days with 25 nM staurosporine before the start of the 48 hour experiment. As a positive control, the differentiating effect of 25nM staurosporine is shown. The neurite length is calculated on at least 5000 cells. Data are provided from the experiments performed in duplicate. Means and standard deviations are shown. Figures 8 to 18 are graphical representations of the effect of enovin on the proliferation of various cell types. Figure 19 is a graphical representation of the effects of enovin on taxol-induced sensory deficits using the pinprick test. Given are the mean (+ 1 SEM) cumulative scores over time in rats treated with either 2 different doses of enovin (23 or 130 ug/ml; n = 10 rats/group) or vehicle/saline (n = 20 rats) after rooftop sun. Enovin or saline/vehicle was injected in a volume of 75 µl into the subplantar area of the right hind paw. Figure 20 is a graphical representation of the effects of enovin on taxol-induced sensory deficits using the pinprick test. Shown are the mean (+ 1 SEM) cumulative scores over time for rats treated with either 2 different doses of enovin (23 or 130 ug/ml; n = 10 rats/group) or vehicle/saline (n = 20 rats) before rooftop sun. Enovin or saline/vehicle was injected in a volume of 75 µl into the subplantar area of the right hind paw. Figure 21 is a DNA sequence of enovin. The consensus sequence was obtained by amplification by PCR using the primers PNHsp5 and PNHapl on cDNA from human frontal cortex and on human genomic DNA followed by cloning, sequence analysis and comparison of the reaction sequences. The predicted amino acid sequence is shown above the DNE sequence for the only splice variant that yields a functional Enovin protein after translation. The nucleotide residue number is shown to the left of the DNA sequence, while the amino acid residue number is shown to the right of the translated protein sequence. 5- and 3' splice sites detected by comparison of sequenced cDNA fragments with the genomic sequence are indicated by vertical lines that bend to the left and right, respectively, and are numbered consecutively. The putative RXXR-furin cleavage site for the prodomains is indicated in bold and underlined. The presumed beginning of the fully developed protein is indicated by an arrow. The seven conserved cysteine residues characteristic of all members of the TGF-15 family are indicated in bold. A potential N-linked glycosylation site is double underlined. 5' and 3' splice sites are numbered and encircled. Figure 22 is an illustration of the expression of different Enovin splice variants in human tissue. (A) schematic diagram of Enovin splice variants identified by RT-PCR experiments with Enovin's specific primers on RNA derived from different human tissues followed by cloning and sequence analysis of PCR products. The top line shows a scale (in bp). The second line represents the enovine genomic sequence. The position of the translation initiation and stop codons for the beginning of the fully developed Enovin coding sequence and of the 5' and 3' splice sites (see Figure 21) are indicated. The right part of the figure shows the PCR products obtained by RT-PCR on ovarian and frontal cortex RNA together with a 100 bp DNA ladder. The position of the different mRNA variants is indicated along with their size (from start to stop codon). The translated proteins are shown on the left. Squares depict regions represented in the cDNA. Dashed lines represent spliced genomic DNA. The shaded area represents the fully evolved enovin coding sequence. The dashed line indicates the beginning of the fully developed enovin coding sequence. The two transcripts capable of yielding functional Enovin protein are indicated by an asterisk on the left. (B) The tissue distribution of the major splice variants. The photograph shows the PCR fragments obtained by RT-PCR with Enovin-specific primers on different human cDNAs. The 4 main splice variants (A to D) are indicated by arrows on the left. The sizes are indicated on the right-hand side based on the 100 bp DNA ladder used as a size reference on the gel. Figure 23: Predicted protein sequence of the long splice variant of Enovin, obtained by splicing out the two introns from the DNE sequence in Figure 21. The splice sites 5'-l and 3'-l are used to remove the first intron, and the splice sites 5'- 2 and 3'-3 are used to remove the second intron. This results in a cDNA sequence having an open reading frame encoding the 228 amino acid residue protein shown above. Figure 24: Predicted protein sequence of an alternative (short) splice variant of Enovin, obtained by splicing out the two introns from the DNE sequence in Figure 21. The splice sites 5'-1 and 3'-2 are used to remove the first intron and the splice sites 5 '-2 and 3'-3 are used to remove the second intron. This results in a cDNA sequence having an open reading frame encoding the 220 amino acid residue protein shown above. This protein sequence lacks 8 amino acid residues compared to the sequence of Figure 23. Figure 25 is a graphical representation of the results obtained from experiments designed to compare the expression levels of enovin in normal diseased tissue. Enovin and GAPDH expression is represented in brain tissue, with respect to multiple sclerosis and Alzheimer's disease. Figure 26 is a graphical presentation of the results obtained by detecting expression levels of enovin and GAPDH in Parkinson's disease and cancer.

Depositions

Plasmid EVNmat/pRSETB including the DNE sequence encoding enovin was deposited on 6 May 1999 under accession no. LMBP3931, at "the Belgian Coordinated Collections of Micro-Biologie" (BCCM) at "Laboratorium voor Moleculaire - Plasmidencollectie" (LMBP) B9000, Ghent, Belgium, in accordance with the regulations set forth in the Budapest Treaty of April 28, 1997.

Materials and methods

Materials

Native Taq polymerase, ampicillin, IPTG (isopropyl-β-D-thiogalactoside), X-gal (5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside) and all restriction enzymes used were from Boehringer Mannheim (Mannhein, Germany ). 10 mM dNTP mix was purchased from Life Technologies (Gaithersburg, MD, USA). The TOPO-TA cloning kit was purchased from Invitrogen BV (Leek, The Netherlands). The Qiagen plasmid mini or midi DNA purification kit, the Qiaprep-Spin-Miniprep kit and the Qiaquick gel extraction kit were purchased from Qiagen GmbH (Dusseldorf, Germany). c DNA collection, Marathon<1>"<1>Ready-cDNA kit, human multiple tissue cDNA (MTC™) lanes I and II "multiple tissue northern blots" and "the Advantage-GC cDNA PCR kit" was acquired from Clontech Laboratories (Palo Alto, CA, USA). All PCR reactions were performed in a "GeneAmp PCR system 9600 cycler" (Perkin Eimer, Foster City, CA, USA). LB medium (Luria-Bertani) consists of 10 g/l tryptone, 5 g/l yeast extract and 10 g/l NaCl 2x YT/ampicillin plates consist of 16 g/l tryptone, 10 g/l yeast extract, 5 g/l NaCl, 15 g/l agar and 100 mg/l ampicillin.

Database homology search and sequence comparison.

Using the complete human glial cell line-derived neurotrophic factor (GDNF; accession no. Q99748), neurturin (NTN; accession no. P39905) and persephin (PSP; accession no. AF040962) cDNA-derived protein sequences as search sequences, a "BLAST" search was (Basic Local Alignment Search Tool; Altschul et al., 1990) performed on the daily update of EMBL/GenBank's human expressed sequence tag (EST) and genomic databases.

Additional BLAST searches were performed using the genomic sequence of accession no. AC005038, and several ESTs occurring in the GenBank database, which showed homology to this genomic sequence, were detected.

The percent identity and percent similarity between members of the GDNF family were calculated by pairwise comparison of sequences using the BESTFIT program (Genetics Computer Group sequence-analysis software package, version 8.0, University of Wisconsin, Madison, WI, USA). The groupings of the DNA or protein sequences were performed with the ClustalW grouping program (EMBL, Heidelberg, Germany).

Oligonucleotide synthesis for PCR and DNA sequencing.

All oligonucleotide primers were ordered from Eurogentec (Seraing, Belgium). Insertion-specific sequencing primers (15- and 16-mers) and primers for use in PCR reactions were developed manually. DNA was prepared on Qiagen tip 20 or 100 anion exchange or Qiaquick spin columns (Qiagen GmbH, Dusseldorf, Germany) and extracted from the columns in 30 µl TE buffer (10 mM Tris.HCl, 1 mM EDTA ( sodium salt), pH 8.0).

The sequencing reactions were performed on both strands using the ABI prism BigDye Terminator Cycle sequencing kit and were run on an Applied Biosystems 377XL sequencing machine (Perkin Eimer, ABI Division, Foster City, CA, USA). Sequencher™ software was used for sequence assembly and manual editing (GeneCodes, AnnArbor, MI, USA).

Cloning of a new GDNF homologue.

A DNA region spanning nucleotides 67411 to 68343 of EMBL accession no. AC005038, the translated protein sequence of which was homologous to fully developed NTN and PSP, was used to develop the oligonucleotide primers for PCR amplification. The different primers that were used are shown in table 1.

The primers PNHsp3 and PNHapl were used to amplify a fragment of 502 bp on cDNA derived from different human tissues (fetal brain, whole fetus, prostate or lung, Marathon-Ready™ cDNA (Clontech Laboratories), frontal cortex, hippocampus and cerebellum) cDNA) and on human genomic DNA. Based on the genomic sequence from the EMBL/GenBank database (acc. no. AC005038), the fragment to be amplified was predicted to have a G+C content of 76%. Therefore, the amplifications were performed using "the Advantage-GC cDNA PCR kit" (Clontech Laboratories, Palo Alto, CA, USA), optimized for the amplification of GC-rich DNA sequences. PCR reactions were performed in a total volume of 50 µl, containing lx GC cDNA PCR reaction buffer, 0.2 mM dNTP, 1 M GC-MELT™, 200 nM of primers PNHsp3 and PNHapl, 1 µl of "Advantage KlenTaq polymerase mix and 1 to 5 µl of cDNA or 0.5 µg of genomic DNA. The probes were heated to 95°C for 5 min, and cyclization was performed for 45

s at 95°C, 1 min at 58°C and 40 s at 72°C for 35 cycles, with a final step of 7 min at 72°C. The probes were finally treated with 2.5 U of native Taq DNA polymerase to add an A overhang. The PCR products were analyzed on a 1%

(w/v) agarose gel in lx TAE buffer (40 mM Tris-acetate, 1 mM EDTA (sodium salt), pH 8.3). PCR fragments of the expected size (495 bp) were removed from the gel and purified with the Qiaquick gel extraction kit. The PCR fragments were sequenced to confirm their identity and cloned into the plasmid vector pCR2.1-TOPO using the TOPO TA cloning

the equipment according to the manufacturer's instructions. Approximately 20 ng of purified fragment was combined with 1 µl of pCR2.1-TOPO vector in a total volume of 5 µl. The reaction mixtures were incubated at room temperature (20°C) for 5 min. 2 µl of the reaction mixture was transformed into TOP10F'-competent cells (Invitrogen BV) using heat-shock transformation and plated on 2x YT/ampicillin plates supplemented with 10 mM IPTG and 2% (w/v) X- crazy for blue-white classification. White colonies after overnight growth were picked from the plates, grown in 5 ml LB medium supplemented with 100 mg/l ampicillin and plasmid DNA prepared using the Qiaprep Spin Miniprep kit. The presence of an insert of the expected size was confirmed by restriction digestion with EcoRI. The plasmid insert of several positive clones was sequenced and the sequences obtained were compared using the Clustal alignment program.

To obtain additional coding sequence for the new GDNF homolog, a fragment with an expected size of 931 bp based on the EMBL/GenBank sequence (accession no. AC005038) was amplified by PCR using the primers PNHspl and PNHapl. PCR reactions were performed in a total volume of 50 µl, containing lx GC cDNA PCR reaction buffer, 0.2 mM dNTP, 1 M GC-MELT™, 200 nM of primers PNHspl and PNHapl,

1 µl of "Advantage KlenTaq" polymerase mix and 1 to 5 µl cDNA from cerebellum, frontal cortex or hippocampus or 0.5 µg of genomic DNA. Probes were heated to 95°C for 5 min and cycling was performed for 45 s at 95°C, 1 min at 58°C and 1 min 30 s at 72°C for 35 cycles, with a final step of 7 min at 72°C. The PCR products were analyzed on a 1% (w/v) agarose gel in lx TAE buffer (40 mM Tris-acetate, 1 mM EDTA (sodium salt), pH 8.3). A second round of amplification was performed with the nested primers (PNHsp2 and PNHap2). 1 µl of the first round amplification reaction was used in a total volume of 50 µl, containing lx GC cDNA-PCR reaction buffer, 0.2 mM dNTP, 1 M GC-MELT™, 200 nM of the primers PNHsp2 and PNHap2 and 1 µl of "Advantage KlenTaq" polymerase mix. The probes were heated to 95°C for 5 min, and cycling was performed for 45 s at 95°C, 1 min at 58°C and 1 min 30 s at 72°C for 35 cycles, with a final step of 7 min at 72°C. The probes were analyzed on a 1% (w/v) agarose gel in lx TAE buffer. PCR fragments of the expected size (870 bp) were removed from the gel and purified with the Qiaquick gel extraction kit. The PCR fragments were sequenced to confirm their identity, treated with 2.5 U of Taq polymerase and cloned into the plasmid vector pCR2.1-T0P0 using the TOPO TA cloning kit according to the manufacturer's instructions. Approximately 20 ng of purified fragment was combined with 1 µl of pCR2.1-TOPO vector in a total volume of 5 µl. The reaction mixtures were incubated at room temperature (20°C) for 5 min. 2 µl of the reaction mixtures were transformed into TOP10-competent cells using heat-shock transformation and were plated on 2x YT/ampicillin plates supplemented with 10 mM IPTG and 2% (w/v) X-gal for blue-white grading. White colonies after overnight growth were picked from the plates, grown in 5 ml LB medium supplemented with 100 mg/l ampicillin and plasmid DNA prepared using Qiaprep Spin Miniprep equipment. The presence of an insert of the expected size was confirmed by restriction digestion with EcoRI. The plasmid insert of several positive clones was sequenced and the sequences were compared using the ClustalW alignment program.

Analysis of enovin gene expression by RT-PCR analysis.

Oligonucleotide primers PNHsp3 and PNHapl (see Table 1) were used for the specific PCR amplification of a 502 bp fragment from enovin. PCR amplifications were performed on human multiple tissue cDNA panels (MTC™) normalized to the mRNA expression levels of six different housekeeping genes. PCR reactions with enovin-specific primers were performed in a total volume of 50 µl, containing 5 µl of cDNA, 1x GC cDNA PCR reaction buffer, 0.2 mM dNTP, 1 M GC-MELT TM, 400 nM of the primers PNHsp3 and PNHapl and 1 µl of Advantage KlenTaq Polymerase Mix. The probes were heated to 95°C for 30 s, and cycling was performed for 30 s at 95°C and 30 s at 68°C for 35 cycles. The probes were analyzed on a 1.2% (w/v) agarose gel in lx TAE buffer (40 mM Tris-acetate, 1 mM EDTA (sodium salt), pH 8.3) and images of the ethidium bromide-stained gels were obtained using an "Eagle Eye II Video" system (Stratagene, La Jolla, CA,

USA).

Similarity searches of the daily update of the EMBL/GenBank database with the human neurturin and persephin protein sequences yielded a genomic DNA sequence encoding a hypothetical new protein similar to the neurotrophic factors GDNF, NTN and PSP called enovin (EVN). Further database homology searches using the genomic DNA sequence surrounding the region encoding enovin yielded several expressed sequence tag (EST) clones derived from different human tissues (prostate epithelium [accession no. AA533512 (ID1322952) ], lung carcinoma [accession no. AA931637] and parathyroid tumor [accession no. AA844072]). These clones contain a DNA sequence outside the region of homology with GDNF, PSP or NTN, but confirmed that enovin mRNA is expressed in normal tissue and in tumor tissue.

Initial PCR amplification using the primers (PNHsp3 and PNHapl) based on the genomic sequence yielded a 500 bp fragment from fetus, fetal brain, prostate, frontal cortex, hippocampus, cerebellum cDNA and from genomic DNA, but not from lung -cDNA. Cloning and sequence analysis of these fragments yielded a DNA sequence of 474 bp that translated into a predicted protein sequence of 139 amino acid residues including seven conserved cysteine residues characteristic of all members of the transforming growth factor β (TGF-β) family of proteins (Kingsley, 1994) (Figure 1). The sequence also contained a RXXR motif for cleavage of the prodomains (RAAR, amino acid positions 23 to 26) (Barr, 1991). A similar cleavage site exists in the GDNF, NTN and PSP protein sequences, at a comparable position in the prodomain sequences. Assuming that cleavage of the enovin prodomain occurs at this site in vivo, the fully developed EVN protein sequence contains 113 amino acid residues (residues 27 to 139 in Figure 1) and has a calculated molecular weight of 11965 Da and an isoelectric point of 11.8. There is one potential N-glycosylation site present in the fully developed sequence (NST at amino acid position 121-123). Furthermore, several conserved regions between the fully developed forms of the known neurotrophic factors GDNF, NTN and PSP were also present in enovin (Figure 2). Table 2 summarizes the results of the comparison of the fully developed protein sequences for GDNF family members using the BESTFIT program. Percent identity and percent similarity are shown. The fully developed GDNF, NTN, PSP and EVN sequences used in this comparison began at the first amino acid residue after the RXXR cleavage site.

From these comparisons, it appears that the fully developed enovin protein is more closely related to persephin and neurturin than to GDNF.

Amplification, cloning and sequence analysis of a larger fragment of the enovin DNA sequence from the frontal cortex cDNA using the primers based on the EMBL/GenBank genomic sequence (accession no. AC005038) yielded a sequence of 819 bp (Figure 3) . This sequence contains an imaginary ATG start codon at nucleotide positions 30-32 and provides an open reading frame (reading frame A in Figure 3) which extends up to a stop codon at nucleotide positions 285-287. The translated protein sequence in this area shows no similarity to any known protein in the databases. Translation of the cDNA' sequence in the second reading frame (reading frame B in Figure 3) gives a predicted protein sequence of 159 amino acid residues. This sequence contains the RXXR cleavage site (position B43 to B46; nucleotide position 460-471) and the sequence corresponding to the fully developed enovin sequence (position B47 to B159; nucleotide position 472-810). The open reading frame including the RXXR cleavage site and the fully developed enovine coding sequence extends from nucleotide position 334 (preceded by an in-frame stop codon) to a stop codon at position 811-813, but does not contain an ATG codon as an outgoing methionine residue . In analogy with persephin (Milbrandt et al., 1998), we hypothesize that an unspliced intron is present in most mRNA transcripts from the EVN gene. GDNF and NTN also have an intron in their respective prodomain coding regions (Matsushita et al., 1997, Heuckeroth et al., 1997).

To assess the presence of different mRNA transcripts for Enovin, RT-PCR experiments were performed using the primers located at the 5' end of the Enovin coding sequence, just 5' of a possible upstream ATG start codon (primer PNHsp5 [ 5'-GCA AGC TGC CTC AAC AGG AGG G-3'] and stranded primer PNHsp6 [5'-GGT GGG GGA ACA GCT CAA CAA TGG-3'] and at the 3' end (primer PNHapl and stranded primer PNHap2 [see table 1]. Experiments were performed on human multiple tissue cDNA arrays (Clontech MTC arrays I and II), on a fetal heart cDNA pool (Clontech), and on cDNA derived from human cerebellum, hippocampus, or frontal cortex (Masure et al., 1998).Primary PCR reactions were performed with primers PNHsp5 and PNHapl under GC-rich conditions (Advantage GC-PCR kit, Clontech) for 30 cycles (95°C - 30s, 60°C - 30s, 72° C - 1 min) as described Nested PCR reactions were performed on 1 µl of the primary PCR product using the primers PNHsp6 and PNHap2 under the same conditions in 30 cycles. Resulting PCR products were analyzed on a 1.5% agarose gel and ranged in size from +350 bp to +800 bp. Several bands were purified from the gel, and the PCR fragments were sequenced directly. Some purified PCR products were also cloned into vector pCR2.1-TOPO (TOPO-TA cloning kit, Invitrogen) and then sequenced. Sequence analysis confirmed the presence of different mRNA molecules containing an Enovin sequence. The obtained fragment sequences were compared with the genomic Enovin sequence. This enabled us to identify several possible 5' and 3' splice sites in the genomic sequence (Figure 21). All these splice sites correspond to the consensus sequences of the donor and acceptor splice sites (Senapathy, P., Shapiro, M.B. & Harris, N.L.

(1990)), the splice sites, branch point sites, and exons: sequence statistics, identification, and genome project applications. Methods Enzymol. 183,252-278). The different Enovin splice variants identified and their presence in different human tissues are summarized in Figure 22. Only two of the 5 sequenced transcripts yield functional Enovin protein when translated from the ATG start codon. These two transcripts encode proteins of 228 and 220 amino acids, respectively, with predicted signal peptides of 47 and 39 amino acid residues. The predicted protein sequences of these two variants are shown in Figure 23 (long variant) and Figure 24 (short variant). The long variant can be derived from the DNE sequence in Figure 21 by splicing the first intron at the locations 5'-1 and 3'-1 and the second intron at 5'-2 and 3'-3. By translating the open reading frame, the predicted protein sequence in Figure 23 is obtained. The shorter variant can be derived from the DNE sequence in Figure 21 by splicing the first intron at the locations

5'-1 and 3'-2 and the second intron at 5'-2 and 3'-3. When translating the open reading frame, the predicted protein sequence in Figure 24 is obtained.

The longest transcript appears to be the most abundant in most tissues judging by the band intensity in Figure 22B. The shorter transcripts result in frameshifts yielding a translated protein lacking the fully developed Enovin amino acid sequence homologous to GDNF, NTN and PSP. The two smallest transcripts even lack part of the fully developed coding sequence, including two of the seven highly conserved cysteine residues. Figure 22B shows the distribution of the main splice variants in different human tissues. Functional Enovin mRNA is expressed in nearly all tissues tested, including brain, heart, kidney, liver, lung, pancreas, skeletal muscle, colon, small intestine, peripheral blood leukocytes, spleen, thymus, prostate, testis, ovary, placenta, and fetal heart. In some human tissues (eg cerebellum, hippocampus), only non-functional transcripts will be able to be amplified by PCR. To our knowledge, the occurrence of non-functional mRNA transcripts has not been described previously to such a large extent. The biological significance of this finding must be studied. Although the expression of NTN and PSP in different tissues has not been fully characterized, their expression levels appear to be much lower and their expression more restricted to certain tissues (Kotzbauer et al., 1996, Milbrandt et al., 1998).

Recombinant expression of Enovin in E. coli

Construction of an Enovin Expression Plasmid

A 414 bp PCR fragment was amplified from human genomic DNA with primers PNHsp4 and PNHap2 (Table 1) and cloned into vector pCR2.1-T0P0 using TA cloning (Invitrogen). The sequence of the insertion was confirmed by sequence analysis. A clone containing an insertion with the Enovin consensus sequence (clone 36) was used for subsequent construction of an expression plasmid. Two primers were designed containing appropriate restriction sites at their 5' ends. The forward primer PNHexp-spl (5'- GC G GAT CCG GCT GGG GGC CCG GGC A -3') contained a BamHI restriction site (underlined) and the reverse primer PNHexp-apl (5'- GCC TCG AGT CAG CCC AGG CAG CCG CAG G -3') contained an Xhol restriction site (also underlined). Using these primers, the 343 bp fragment encoding fully developed Enovin (position 81 to 422 in Figure 1) was amplified from clone 36. The PCR reaction was performed in a total volume of 50 µl, containing 1x GC cDNA PCR reaction buffer , 0.2 mM dNTP, 1 M GC-MELT™, 200 nM of primers PNHexp-spl and PNHexp-apl, 1 µl Advantage KlenTaq polymerase mix and 10 ng plasmid DNA from clone 36. The probes were heated to 94°C in 5 min, and cycling was performed for 45 s at 94°C, 1 min at 58°C and 30 s at 72°C for 25 cycles, with a final step of 7 min at 72°C. The resulting 50 µl product was purified using the Qiaquick PCR purification kit (Qiagen), and DNA was eluted in 30 µl. 25 µl of this purified product was then digested in a 30 µl reaction with 10 U of BamHI and 10 U of XhoI in 1x buffer B (Boehringer Mannheim) for 1 h at 37°C. After electrophoresis in a 1% (w/v) agarose gel in lx TAE buffer (40 mM Tris-acetate, 1 mM EDTA (sodium salt), pH 8.3), the expected 353 bp band was excised from the gel and purified using Qiaquick Gel Extraction Kit. The resulting fragment was ligated into the vector pRSET B (Invitrogen) linearized with BamHI and XhoI. The insertion of the resulting plasmid assembly (hEVNmat/pRSETB) was confirmed by complete sequence analysis. The resulting construct encodes a 14 6 amino acid protein with a predicted molecular weight of 15704 Da including an NH2-terminal 6x His-tag fused in-frame to the full-length enovin coding sequence. Thus, the NH2-terminal amino acid sequence of the resulting protein is MRGS HHHHHHGMASMTGGQQMGRDLYDDDDKDPAGGPGS (fully developed Enovin sequence in bold, 6x His-tag underlined).

Expression of Enovin in BL21(DE3) E. coli cells

Recombinant production of Enovin protein was performed essentially as described for Neurturin by Creedon et al.

(1997), with modifications. For the production of recombinant Enovin protein, plasmid hEVNmat/pRSETB was transformed into an E. coli strain BL21(DE3) (Novagen) and grown in 2xYT/ampicillin medium (16 g/l tryptone, 10 g/l yeast extract, 5 g/ l NaCl and 100 mg/l ampicillin) at 30°C (225 rpm) or 37°C (300 rpm) to an OD600 of approximately 0.5 before addition of IPTG to a final concentration of 0.2 mM to induce expression. Cell pellets were harvested by centrifugation after a 3 hour induction period, washed with phosphate buffered saline, centrifuged and stored frozen. For purification and refolding, cell pellets were resuspended in sonication buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM 2-mercaptoethanol, protease inhibitors

(Complete TM protease inhibitor cocktail tablets (Boehringer Mannheim, 1 tablet per 50 ml buffer) and 1 mg lysozyme per 500 mg cell pellet). The cells were disrupted by sonication, and the inclusions were harvested by centrifugation. The inclusions were dissolved and incubated in buffer A (8 M urea, 20 mM Tris-HCl pH 7.6, 200 mM NaCl, 1 mM 2-mercaptoethanol) for 30 min at 37°C before addition to Ni-NTA resin (nickel nitrilotriacetic acid, Qiagen ). After 40 min of shaking at 37°C, the probes were washed once with buffer A and loaded onto a 5 ml Ni-NTA column. The column was washed successively with 10 column volumes of buffer A, 10 column volumes of buffer A at pH 7.2 and 10 column volumes of buffer A at pH 7.2 + 10 mM imidazole. The enovin was eluted from the column in 4 column volumes of buffer A at pH 7.2 + 200 mM imidazole.

Enovin refolding was performed by stepwise dialysis overnight at 4°C in renaturation buffer (0.1M sodium phosphate, 0.15M NaCl, 3uM cysteine, 0.02% Tween-20, 10% glycerol, 0.01M Tris-HCl, pH 8.3) containing decreasing amounts of urea at each step (6M to 4M to 3M to 2M to IM to 0.5M to OM urea). The purified protein was aliquoted, stored at -20°C and further used for functional assays.

Chromosomal localization of the Enovin gene.

A 3.3 kb fragment of the Enovin gene was amplified from cerebellum cDNA using the primers EVN(7)-spl (5'- TTC GCG TGT CTA CAA ACT CAA CTC CC -3') and PNHapl (5'- GCA GGA AGA GCC ACC GGT AAG G -3') developed on the sequence of EMBL accession number AC005038. The PCR reaction was performed in a total volume of 50 µl, containing lx Expand Long Template PCR reaction buffer (Boehringer Mannheim), 0.5 mM dNTP, 1 M GC-MELT( (Clontech Laboratories), 400 nM of the primers EVN(7 )-spl and PNHapl and 1 μl of cerebellum cDNA. After an initial 2 min at 94°C, 0.75 μl of Expand Long Template polymerase (Boehringer Mannheim) was added, and cyclization was performed for 10 s at 94°C, 30 s at 58°C and 3 min at 68°C for 10 cycles. Then 20 additional cycles were performed increasing the extension time at 68°C by 20 s each cycle. A final 7 min at 68°C was also included. The resulting 3.3 kb fragment was purified after electrophoresis in a 0.8% agarose/TAE gel and cloned into vector pCR2.1-TOPO using TA cloning (Invitrogen).Complete sequence analysis of the 3.3 kb insert of one clone confirmed that the cDNA sequence obtained corresponded to the genomic sequence in the EMBL database (accession number AC005038). No introns were spliced in the cDNA obtained from the cerebellum m-cDNA.

Chromosomal mapping studies were performed using fluorescent in situ hybridization (FISH) analysis

mainly as described (Heng et al., 1992, Heng&Tsui, 1993). Human lymphocytes were cultured at 37°C for 68-72 h before treatment with 0.18 mg/ml 5-bromo-2'-deoxyuridine (BrdU) to synchronize the cell cycles in the cell population. The synchronized cells were washed and recultured at 37°C

for 6 h. The cells were harvested, and slides were prepared using standard procedures including hypotonic treatment, fixation and air drying. The 3.3 kb probe for Enovin was biotinylated and used for FISH detection. The slides were heated at 55°C for 1 h, treated with RNase and denatured in 70% formamide in 2x NaCl/Cit (20x NaCl/Cit is 3 M NaCl, 0.3 M disodium citrate, pH 7.0) for 2 min at 70°C followed by dehydration with ethanol. The probe was denatured before being applied to the denatured chromosome preparations. After overnight hybridization, the preparations were washed, and FISH signals and the 4',6-diamidino-2-phenylindole banding pattern were recorded separately on photographic film, and the parameters of the FISH mapping data with chromosomal bands were obtained by superimposition of FISH signals with 4',6-diamidino-2-phenylindole-banded chromosomes (Heng&Tsui, 1993). Under the conditions used, hybridization efficiency was approximately 72% for this probe (among 100 mitotic figures examined, 72 of them showed signals on a pair of chromosomes). Since the 4',6-diamidino-2-phenylindole banding was used to identify the specific chromosome, the parameter between the signal from the probe and the short arm of chromosome 1 was obtained. The detailed position was further determined based on the sum from 10 photographs (Figure 4A). There was no additional location selected by FISH detection under the conditions used, therefore we conclude that Enovin is located on human chromosome 1, the region p31.3-p32. An example of the mapping results is shown in Figure 4B.

From the gene mapping data at the National Center for Biotechnology Information (NCBI,

http://www.ncbi.nlm.nih.gov/genemap), it can be deduced that the genomic clone containing the Enovin sequence (EMBL accession number AC005038) is located on chromosome 1, between markers D1S2843 and D1S417. This corresponds to chromosome 1, region p31.1 to p32.3, confirming the data obtained by FISH analysis.

Tissue distribution of Enovin determined by Northern blot and dot blot analysis.

"Northern blot" samples containing 2 µg of poly(A)-rich RNA derived from various human tissues (Clontech Laboratories, Palo Alto, CA, USA; MTN™ blot, MTN™ blot II and Fetal MTN™ blot II) were hybridised according to the manufacturer's instructions with a (α-<32>P-dCTP-random-priming labeled (HighPrime kit, Boehringer Mannheim) 897 bp Enovin fragment. This fragment was obtained by PCR amplification with the primers PNHspl and PNHapl on the frontal cortex -cDNA and subsequent cloning into the vector pCR2.1-T0P0 The fragment contains 897 bp Enovin sequence up to the stop codon and comprises the complete coding sequence for the fully developed Enovin protein.

Enovin mRNA was detected as a major transcript of approximately 4.5 kb (Fig. 5A-C). Enovin mRNA was expressed in several different tissues, primarily in heart, skeletal muscle, pancreas and prostate. Some minor transcripts occur in e.g. placenta (4 kb, 2.4 kb and 1.6 kb) and prostate (4 kb and 1.6 kb). In fetal tissue, a dominant 2.4 kb transcript occurs in liver and to a lesser extent in lung. Other transcripts are also present in fetal kidney, liver, lung and brain.

In addition, an RNA master blot (Clontech Laboratories) containing poly(A)-rich RNA from various human tissues and developmental stages was also hybridized with the 897 bp Enovin probe. The poly(A-rich RNA samples used to prepare this blot have been normalized to the mRNA expression levels of eight different housekeeping genes by the manufacturer. Enovin mRNA was universally expressed, but the highest mRNA levels were found to be in the prostate, the pituitary gland, trachea, placenta, fetal lung, pancreas and kidney (Figure 5D+E) .

Construction of GFRα-IgG-Fc fusion vectors

cDNA regions of GFRα-1, GFRα-2 and GFRα-3 (coding for amino acids 27 to 427, 20 to 431 and 28 to 371, respectively) excluding sequences coding for the signal peptide and for the COOH-terminal hydrophobic region involved in GPI anchoring, was cloned in-frame into the expression vector Signal plg-plus (R&D Systems Europe Ltd). The resulting protein expressed from these constructs contains a 17 amino acid NH2-terminal CD33 signal peptide, GFRα protein region and a 243 amino acid COOH-terminal human IgGi-Fc fusion domain. CHO cells were transfected with GFRα fusion constructs, and stably transfected cells were selected using 500 µg G418. Permanent clones were selected using anti-Fc antibody. For purification of GFRα fusion proteins, cells were grown in serum-free medium, and medium was collected after every 3 days. The medium was centrifuged and transferred to a protein A column (Protein A Sepharose, Pharmacia Biotech). Bound protein was eluted with 0.1 M Na citrate, pH 3.0 and collected in 1 M Tris buffer, pH 8.4. Protein concentration was determined by absorbance at 280 nm using an extinction coefficient of 1.5. These purified soluble GFRα-1 to -3 Fc fusion proteins were used for subsequent binding studies.

Resonance analysis of surface plasmon

Surface plasmon resonance (SPR) experiments were performed at 25°C using a BIAcore 3000 instrument. Assays were performed with enovin and NGF as immobilized ligands. The carboxylated matrix of a F1 sensor chip was first activated with a 1:1 mixture of 400 mM N-ethyl-N-(dimethylaminopropyl)carbodiimide and 100 mM N-hydroxysuccinimide for 10 min. Then, recombinant enovin and NGF were applied to the activated surface in 10 mM acetate buffer, pH 4.5 at a flow rate of 5 µl/min. Vacant groups were inactivated with 1 M ethanolamine hydrochloride. For the binding experiments, soluble GFRα1-3-Fc was superfused at concentrations of 10-100 nM in HEPES-buffered saline (150 mM NaCl, 3.5 mM EDTA, 0.05% P-20, 10 mM HEPES, pH 7, 4) at a flow rate of 10^1/min. Association was monitored for 3 min, and dissociation for 1 min, followed by regeneration with 5 mM NaOH. The dissociation was initiated by superfusion with HEPES-buffered saline. A BIAcore evaluation software, 3.0 was used to calculate the association rate (ka), the dissociation rate (kd) and the equilibrium dissociation constants (KD, calculated as kd/ka).

Results

SPR was used to measure the binding of soluble GFRα1-3 to immobilized enovin. Specific binding to enovin could be detected with only the soluble GFRα3. GFRα1 and GFRα2 were not bound to the immobilized enovin. The observed binding of GFRα3 was specific in that there was no binding to NGF. In the separate control experiment, specific binding of TrkA-Fc (NGF receptor) to the immobilized NGF was detected without binding to immobilized enovin.

From the binding curves obtained using three different concentrations of GFRα, the following constants in Table 3 were derived. These results show that GFRα3 binds specifically to enovin.

Since GDNF, NTN, and PSP all promote the maintenance and survival of different types of neuronal cells, enovin is thought to have similar biological effects on nerve cells and possibly on other cell types as well. Therefore, it is conceivable that the enovin protein may be useful in the treatment of neural disorders in general, including Parkinson's disease, Alzheimer's disease, peripheral neuropathy, amyotrophic lateral sclerosis (ALS), Huntington's disease, acute brain injury, nervous system tumors, multiple sclerosis, peripheral nerve trauma or exposure injury for neurotoxins.

Enovin may also be useful in various aspects of neuroprotection. Considering its effect on the survival of different neuronal cell populations and on the observed neurite extensions in our model of SHSY5Y cells, we hypothesize that this compound may have neuroprotective and neuroregenerative applications.

This is based on the following observations. Taxol induces neuronal apoptosis in NGF-differentiated rat PC12 pheochromocytoma cells (Nuydens et al, submitted). Therefore, taxol-induced cytotoxicity has features of neuronal apoptosis, as monitored by DNA fragmentation, Annexin V labeling and bcl-2 protection. In a broader sense, it can therefore be deduced that taxol induces apoptosis in differentiated SH-SY5Y cells. Enovin has now been shown to be able to reduce this cell death and can therefore reverse neuronal apoptosis in general.

The compound may therefore be useful in the following neurodegenerative conditions where apoptosis has been observed, stroke (Hakim 1998), Parkinson's disease (Marsden et al 1998), Alzheimer's disease (Nagy et al 1998), Huntington's disease (Wellington et al. 1997), neurotrauma (Smirnova et al. 1998), peripheral neuropathies, (Srinivisan et al. 1998).

As an example of the latter clinical indication, we have shown that this neurotrophic factor actually protects differentiated human SH-SY5Y neuroblastoma cells against taxol-induced cell toxicity.

Methodology for measurements of viability

Cell viability was determined by adding 100 μl of a 1 mg/ml 2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide solution (XTT, Sigma) in DMEM ( 37°C) supplemented with 0.02 mM phenazine methosulfate (PMS, Sigma) to each well. The plates were then incubated at 37°C for 2.5 hours. The optical densities were read (Molecular apparatus) at 450 nm, using 650 nm as reference. The XTT assay is based on the conversion of the tetrazolium salt XTT into a red colored formazan product. This reaction is carried out with the help of mitochondrial enzymes.

Methodology for neuronal differentiation

1. Differentiation in human neuroblastoma SHSY5Y cells

SHSY5Y cells are differentiated for 5 days with 25 nM staurosporine. The effect of Enovin is measured 72 hours after the start of the experiment (reference Jalava et al. "Protein Kinase inhibitor staurosporine induces a mature neuronal phenotype in SH-SY5Y human neuroblastoma cells through an a,b,z PKC independent pathway", Journ cell Physiol 155, 301-312

(1993)).

2. Painting of neurite extension.

Morphological changes of neurons were automatically quantified as follows: Briefly, at appropriate times, glutaraldehyde was added directly to the medium which was allowed to stand for 30 min at room temperature. This ensured that the morphology of the cells at that time reflected the real situation. The cells were observed through a transmitting light mode in an Axiovert microscope (Zeiss Oberkochen, Germany), equipped with a Marzhauser scanning stage powered by an Indy workstation (Silicon Graphics, Mountain View, USA). Images were captured using an MX5 video camera (HCS). About. 3000 cells were assessed in 64 aligned images, forming an 8x8 square matrix of images. The precise grouping of the images ensured that the neurites could be followed from one imaging area to the next. Automatic detection of the neurite extensions, labeled with polyclonal tau antibody was performed using an unloaded detector of curvilinear structures (Steger 1998). The analytical software automatically calculated the total cell area, the number of cells and the total neurite length.

To examine the effect of enovin on different cell types, two assays were performed, a DNA synthesis assay and a chemotaxis assay.

DNA synthesis assay

Cells including human dermal fibroblasts (39SK), human umbilical vascular endothelial cells (HUVEC), human smooth muscle cells (HSMC), human chondrocytes and rat osteoblasts were maintained in DMEM containing 10% FBS (39-SK, HSMC, rat osteoblasts) or defined media (chondrocytes and HUVEC) at 37 °C with 5% CO 2 and 95% air. For the DNA synthesis assay, cells were seeded in a 96-well tissue culture plate at a density of 5000 cells/well in DMEM containing 10% FBS and incubated for 24 h. The culture medium was then replaced with DMEM containing different concentrations of Enovin and 0.1 % BSA (for 39-SK, osteoblasts, HSMC, chondrocytes) or DMEM containing different concentrations of Enovin and 0.5% FBS (for HUVEC) and the cells were incubated for 24 h. Then the culture medium was replaced with 100 µl DMEM containing 5% FBS and 0.1 µCi [<3>H]-thymidine. After 2 hours of pulse labeling, the cells were fixed with methanol/acetic acid (3:1, vol/vol) for 1 hour at room temperature. The fixed cells were washed twice with 80% methanol. The cells were dissolved in 0.05% trypsin (100 µl/well) for 30 min and then in 0.5% SDS (100 µl/well) for another 30 min. Aliquots of cell lysates (180 µl) were combined with 2 ml scintillation cocktail, and radioactivity in the cell lysates was measured using a liquid scintillation counter (Wallac 1409).

Chemotaxis assay

The cells were maintained as described in "DNA synthesis assay". The chemotactic activity of Enovin was assayed using a 12-well modified Boyden Chamber (McQuillan, D.J., Handley, C.J., Campbell, M.A., Bolis, S., Milway, V.E., Herington, A.C., (1986), "Stimulation of Proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage", Biochem. J. 240:423-430). The cells were trypsinized using 0.05% trypsin and 0.5 mM EDTA and resuspended in DMEM. To the bottom wells of a Boyden chamber, aliquots of 150 µl of media containing various concentrations of Enovin were added. A polycarbonate membrane (8 µm) coated with 0.1 mg/ml of type I collagen was placed on top of the bottom wells, followed by assembly of the top wells. Aliquots of 100<y>l cells (70000 cells/ml) were added to the top wells. After a 6 hour incubation period, the apparatus was disassembled. Cells remaining on top of the membrane were removed. The membrane was fixed with 10% formaldehyde for 15 min, followed by staining with Gill's strength hematoxylin. The cells were counted by microscopy (250x magnification), and the average number of cell counts from five areas of each well was used. Each experiment was repeated at least four times. The results were expressed as the multiplicity of the control (DMEM containing 0.1% BSA).

As illustrated by the results in Figures 8 to 18, enovin has no effect on the proliferation in each of the cell types used, or on the migration of HUVEC cells (Figure 14) as described above. There was an effect of enovin on SH-SY-5Y neuroblastoma cells. This showed the selective effect of enovin on neuronal cells.

Both GDNF and NTN have been shown to signal via a signaling complex consisting of a ligand-binding subunit, either GFRα-1 or GFRα-2, and a signaling subunit, the cRET protein tyrosine kinase. Enovin is expected to exert its biological effects via a similar signaling complex consisting of a GFRα-binding partner (either GFRα-1, GFRα-2, the recently characterized orphan receptor GFRα-3 or other currently uncharacterized members of the GFRα family) in combination with cRET or another signaling partner. In fact, our binding data show that enovin can bind specifically to GFRα-3.

In humans, germline mutations in GDNF or cRET can lead to several disease phenotypes including multiple endocrine neoplasia and familial Hirschsprung disease (HSCR) (Romeo et al., 1994, Edery et al., 1994, Angrist et al., 1996). Both diseases are associated with intestinal motility, and Hirschsprung's disease is the most common cause of congenital intestinal obstruction in children. Interestingly, GDNF and cRET knockout mice exhibit remarkably similar pathologies with renal agenesis and intestinal aganglionosis (Sanchez et al., 1996; Moore et al., 1996; Pichel et al., 1996). Enovin may be involved in similar disturbances in the gut or kidney or, since it is universally expressed, it may be important in the development of other peripheral organs in the body.

Interaction of ligands with their receptors is generally achieved by the interaction of specific bonds from particular residues in both proteins. Fragments of a protein can serve as agonists that activate the receptor to exert its growth-promoting and survival-preserving effects on cells. Parts of enovin or synthetic peptides based on the enovin protein sequence may therefore be useful as agonists or antagonists to regulate its receptor GFRα3. Using peptide synthesis or recombinant techniques, hybrid growth factors consisting of parts of GDNF, NTN or PSP or any other neurotrophic factor or growth factor with parts of enovin can be prepared to give a new synthetic growth factor with new properties.

Two pilot experiments were conducted to test whether enovin is able to reverse taxol-induced sensory deficits in rats after subplantar injections in rats. In a first experiment, it was tested whether a single treatment with enovin could reverse taxol-induced sensory deficits, while in another experiment it was tested whether enovin could prevent the development of taxol-induced deficits.

Reversal over time of taxol-induced sensory dysfunction.

Procedure

Male Sprague-Dawley rats weighing 300-340 grams were used. The animals were housed individually with food and water ad libitum. Before the start of the experiment, the animals were placed in standard observation cages and after a habituation period of 15 min, the pinprick reflex was evaluated. To do this, the plantar surface of the animal's right paw was stimulated with a needle, and the reactivity to this needle prick was set as either present (score = 1) or absent (score = 0). Within a session, the procedure was repeated three times with an hourly interval of 1 min between 2 consecutive stimulus presentations; itself, the needle stick test consisted of 3 measurements of the reaction to a needle stick. Only rats with normal reactions to 3 needle pricks were included in the experiment.

On 3 consecutive days in the morning, the animals received a daily subplantar injection of 50 ul taxol (3 mg/ml paclitaxel dissolved in cremophor and dehydrated alcohol plus water) in the right hind paw. The next morning, the pinprick reflex was reassessed, and animals that showed no reactivity to the 3 stimulus presentations were selected. These animals were randomly divided into subgroups (n = 10/group) and received a subplantar injection in the right hind paw of 75 µl of either vehicle, saline, or 23 or 130 µg/ml enovin. Because no differences were observed between the results of animals treated with vehicle and saline, the two groups were combined (control group). On days 1, 4, 5 and 7 after the last treatment, the pinprick test was performed both in the morning (between 8 and 9 a.m.) and in the afternoon (between 3:30 p.m. and 4:30 p.m.). On day 8, a final pinprick test was taken during the morning. For each animal, the cumulative score of needle stick reactivity was measured over time. Because a total of 9 pinprick tests (each consisting of 3 pinprick presentations) were performed after the last treatment, the maximum score that can be obtained during the entire time period of the experiment is equal to 27.

Results

Repeated subplantar injections of taxol over 3 consecutive days result in an acute inflammatory reaction with a lack of response to a pinprick stimulation in most animals. A subplantar injection of saline or vehicle did not affect the taxol-induced deficit. At the first measurement, only 4 of 20 controls showed at least 1 reaction to the three pinpricks, and the mean (+ SEM) pinprick score of the controls at the first measurement was 0.25 (+ 0.12); this is in contrast to the beginning of the experiment where the average score was 3.0 (+ 0.0) because all animals responded to the needle prick. Even after 8 days of measurement, the reactivity of the controls was still impaired, and 11 of 20 rats responded at least

once with an average pinprick score of 0.75 (+ 0.18). Within this control group, none of the rats showed a normal reactivity to all 3 stimuli. The cumulative pinprick score in the controls over time is presented in

Figure 19. Because the animals were tested 9 times during an 8-day period, the maximum score that could be obtained with 3 needle pricks in each test is equal to 27. As can be seen from the diagram, a subplantar injection of saline or vehicle was not in able to reverse the taxol-induced deficit during the tested time period. The mean total cumulative score for the controls at the end of the experiment was 5.10 (+ 0.87); which is 18.9% of the maximum score that can be achieved.

A single subplantar injection of 75 µl of 23 µg/ml enovin, after the first measurement, resulted in 4 out of 10 rats responding at least once, with a mean pinprick score of 0.70 (+ 0.33). On day 8, all 10 animals reacted at least once to the needle prick, and a normal reactivity occurred in 5 out of 10 rats. The average number of needle points in this group on day 8 was 2.20 (+ 0.29). Compared to the controls, the mean number of cumulative points at the end of the 8 days of measurement was significantly increased (Mann - Whitney U-test, two-tailed, p < 0.01) and reached a mean pinprick score of 14.50 (+ 1, 96) (Figure 19). This is 53.7% of the maximum score.

Also with a subplantar injection of 130 ug/ml enovin there was improved efficacy compared to the controls. At the first measurement after 130 µg/ml enovin, 6 of 10 rats reacted at least once with a mean pinprick score of 1.10 (+ 0.35). On day 8, all 10 animals responded to at least one needle prick with a mean score of 2.60 (+ 0.22). A normal reaction to the 3 needle pricks occurred in 8 out of 10 rats. The mean total cumulative pinprick score at the end of the experiment in this group was 17.20 (+ 1.94). This is 63.7% of the total possible score and significantly improved compared to the control group (p < 0.01).

Prevention over time of taxol-induced sensory dysfunction.

Procedure

Male Sprague-Dawley rats weighing 300-340 grams were used. The animals were housed individually with food and water ad libitum. Before the start of the experiment, the animals were placed in standard observation cages and after an adaptation period of 15 min, the pinprick reflex was evaluated. To do this, the plantar surface of the animal's right paw was stimulated with a needle, and the reaction to this needle prick was scored as either present (score = 1) or absent (score = 0). Within a session, the procedure was repeated three times with a time interval of 1 min between 2 consecutive stimulus presentations; as such, the needle stick test consisted of 3 measurements of the reaction to a needle stick. Only rats with a normal reaction to the 3 pinpricks were included in the experiment (pinprick score = 3). After this control measurement, the animals were randomly divided into subgroups (n = 10/group), and they received a subplantar injection in the right hind paw of 75 µl of either vehicle, saline or 23 or 130 µg/ml enovin. Because no differences were observed between the results of vehicle- and saline-treated animals, the two groups were combined (control group). During the 3 following days, the animals received a daily subplantar injection of 50 µl taxol (3 mg/ml paclitaxel dissolved in cremophor and dehydrated alcohol plus water) in the right hind paw. On days 1, 4, 5 and 7 after taksol, the pinprick test was performed both in the morning (between 8 and 9 a.m.) and in the afternoon (between 3.30 p.m. and 4.30 p.m.). On day 8, a final pinprick test was performed during the morning. For each animal, the cumulative score for the reaction to the needle prick was measured over time. Because a total of 9 pinprick tests (each consisting of 3 pinprick presentations) were performed after the taxol treatment, the maximum cumulative score that can be obtained during the total time period of the experiment is equal to 27.

Results

A subplantar injection of saline or vehicle before taxol did not prevent the taxol-induced deficit in the pinprick test. At the first post-taxol testing, 8 out of 20 rats responded at least once to the needle stick, with a mean needle stick score of 0.60 (+ 0.18). On day 8, the taxol-induced deficit was still present, and only 8 of 20 animals responded, with a mean score of 0.8 (+0.25). In two animals a normalized pinprick reflex was present. Over time, the cumulative pinprick score also decreased, resulting in a mean value of 6.55 (+ 1.08), which is 24.3% of the maximum score (Figure 20).

Pretreatment with 23 ug/ml enovin reduced the taxol-induced deficits on the pinprick. On day 1, 8 out of 10 animals responded at least once, and the mean pinprick score was 1.70 (+ 0.40). On day 8, all animals responded with an average score of 2.50 (+ 0.27) . Here, 7 animals showed a normal reactivity to all needle pricks. With regard to the cumulative response over time (Figure 20), the average total score was significantly improved (p < 0.01)

relative to the control level of 18.40 (+ 1.73); this is 68.1% of the maximum value.

Comparable results were obtained after a pretreatment with 130 µg/ml enovin. Here, 6 out of 10 animals responded during the first testing with an average pinprick score of 1.70 (+ 0.31). On day 8, all animals responded at least once to a pinprick stimulation with a mean score of 2.40 (+ 0.22), and all 3 responses were normal in half of the animals. With regard to the cumulative score, the average score obtained on day 8 is equal to 17.70 (+ 1.92), which represents 65.5% of the total score.

The present series of experiments indicates that a single subplantar injection of enovin is capable of reducing the taxol-induced sensory deficits measured by a pinprick test. Activity is shown when the drug was administered both before and after taxol.

Enovin is a possible candidate for pain syndromes with mainly a peripheral and central neurogenic component, rheumatic/inflammatory diseases as well as conductance disorders, and may play a modulatory role in sensory processes after transdermal, topical, local, central (such as epidural, intrathecal and similar) and systemic application. Furthermore, it is profitable to use enovin as a diagnostic tool to screen for physiopathological changes in the areas mentioned above.

Comparison of Enovin mRNA expression in normal versus diseased tissue

The expression of Enovin mRNA was quantitatively analyzed using the ABI Prism 7700 sequence detection system (TaqMan; Perkin Eimer) using proprietary technology developed and performed at Pharmagene Laboratories Ltd, Royston, United Kingdom. The system uses a fluorogenic probe to generate sequence-specific fluorescent signals during PCR. The probe is an oligonucleotide with accompanying fluorescent reporter and inactivator dyes, it is placed between the forward and reverse PCR primers. While intact, the intensity of reporter fluorescence is suppressed by the inactivator. Should the probe form part of a replication complex, the fluorescent reporter is cleaved from the inactivator by a 5' to 3' exonuclease activity inherent in Taq polymerase. The increase in fluorescent reporter signal within a reaction is a direct measure of the accumulation of PCR product. The starting copy number of an mRNA target sequence (Cn) is determined by determining the fractional PCR cycle number (Ct) at which a PCR product is first detected B the point at which the fluorescence signal passes above a threshold line. Quantification of the amount of target mRNA in each probe is determined by comparison of experimental Ct values with a standard curve.

RNA preparation and quality control

Total RNA was isolated from whole and sub-dissected tissues, using Tri-Zol reagent (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer's protocol. Quality control procedures for all RNA samples included an assessment of integrity (intact 18S and 28S ribosomal RNA) and determination of the presence of high abundance (actin) and low abundance (transferrin receptor) transcripts.

Primer/probe development

A pair of primers and a TaqMan probe were designed to amplify a specific sequence from Enovin

In addition, a pair of primers and a TaqMan probe were developed that span an intron and amplify part of the human GAPDH gene

Probe 5 is labeled with fluorine-FAM while probe 6 is labeled with fluorine-VIC.

DNase treatment of total RNA

For each tissue tested, 2.2 µg of total RNA was digested with 2 units of RNase-free DNase (Gibco BRL) for 15 min at room temperature in a 20 µl volume of lx DNase buffer (Gibco BRL). The reaction was stopped by the addition of 2 µl of 25 mM EDTA solution. The probes were then incubated at 65°C for 10 minutes to inactivate the enzyme.

Synthesis of first strand cDNA

For each tissue tested, 100 ng of total RNA was used as template for first strand cDNA synthesis. The RNA in a volume of 4 L and in the presence of 50 nM of primers 1 and 2, lxPCR buffer II (Perkin Eimer) and 5 mM MgCl 2 was heated to 72°C for 5 minutes and cooled slowly to 55°C. After addition of all other reagents, 6 L of the reaction mixture was incubated at 48°C for 30 minutes followed by an enzyme inactivation step at 90°C for 5 minutes. The final reaction conditions were as follows: lxPCR buffer II, 5mM MgCl 2 , 1mM dATP, dTTP, dGTP, dCTP, 12.5 units of MuLV reverse transcriptase (Gibco BRL).

PCR amplification of the first chain cDNA products

The cDNA derived from 100 ng of total RNA for each probe was subjected to PCR amplification in a single reaction to identify both the target and GAPDH transcripts. The final primer/probe concentrations for the target transcripts were 300nM primer 1, 300nM primer 3 and 200nM probe 5, for GAPDH they were 20nM primer 2, 20nM primer 4 and 100nM probe 6. The final concentration of other reagents in the reaction was 4, 5% glycerol, 1 x TaqMan buffer A (Perkin Eimer), 6.25mM MgCl2, 430M dATP, dUTP, dGTP, dCTP, 2.5 units AmpliTaq Gold. The PCR amplification was performed in the ABI 7700 sequence detection system, an initial enzyme activation step at 94°C for 12 min was followed by 45 cycles at 94°C for 15 sec, 60°C for 1 min (minimal ramp time).

Diseases and tissues tested

Enovin mRNA expression was compared in tissues taken from diseased patients and normal control subjects (Figures 25 and 26).

The table below shows the diseases and corresponding tissues that have been examined. For each condition, three disease samples and three control samples were analyzed.

Statistical analysis

For each group of 3 tissues, the mean and standard deviation were calculated for the Ct values (which are normally distributed), and they were then converted to Cn values according to the formula Cn = 10(<ct-40- 007>/-3 -623> .analysis of variance (ANOVA) was performed on the Ct values also to compare the mean Enovin mRNA expression levels in normal tissue versus diseased tissue.

Figures 25 and 26 show the average Enovin mRNA copy numbers (+SD; n=3) in diseased tissue compared to control tissue. Statistical analysis showed a significant increase in the Enovin expression level in the periventricular white matter in patients with multiple sclerosis (p = 0.013). The internal GAPDH control showed no significant difference (p = 0.79). Although the Enovin expression level in the periventricular white matter is quite low in normal tissue (270 copies per 100 ng of total RNA on average relative to 200,000 copies of GAPDH), the level is three times higher (825) in patients with multiple sclerosis.

Only one other diseased tissue showed a significant difference compared to the normal control: in ductal breast adenocarcinoma, the Enovin mRNA expression level is 6 times higher (6000 versus 1000; p = 0.007), but the GAPDH control value is also significantly increased (165000 versus to 44000; p = 0.03), and probably represents a general increase in mRNA levels.

In conclusion, we have found that Enovin mRNA levels are upregulated in the periventricular white matter of patients with multiple sclerosis.

Application of cell-based phospho-specific antibody ELISA

for screening of enovin mimetics on 6FRa3/cRET receptor complex.

The method can also be used to identify agonists or antagonists of other neurotrophin receptors, such as GFRa1, GFRa2, GFRa4, TrkA, TrkB and TrkC.

Assay

Using this assay we can identify agonist or antagonist compounds of neurotrophic growth factors by measuring the activation of key signaling kinases activated in the neurotrophic reaction pathway or by measuring the activation of cRET receptor kinase. The activation is measured by detecting the amount of phosphorylated kinase or receptor kinase using phospho-specific antibodies. We will use NIH 3T3 cells transiently or permanently expressing TrkA, TrkB, TrkC, GFRa1/cRET, GFRa2/cRET, GFRa3/cRET or GFRa4/cRET.

The activation of p42/p44 MAP kinase, PKB kinase, c-jun, CREB, JNK/SAPK kinase and other kinase is detected using commercially available phospho-specific antibodies. In addition, cRET activation can be removed using phospho-specific cRET antibody.

The protocol used was as follows:

-Sowed NIH 3T3 cells in 96-wells in 10% calf serum, the cells must be 80% confluent before the stimulation. -The next day, replace the medium with serum-free medium and starve the cells for 18-24 hours. -After starvation, stimulate the cells with compounds and neurotrophic factors as positive control (10 ng/ml for neurotrophic factors) -Fix the cells with 4% formaldehyde in PBS at 4°C for 20 min. -Wash the cells 3x with 200 ul PBS/0.1% Triton for 5 min. - Quench the cells with 100 µl 0.6% H2O2 in PBS/0.1% Triton for 20 min. -Wash the cells 3x with 200 ul PBS/0.1% Triton for 5 min. -Block the cells with 100 µl 10% fetal calf serum in PBS/0.1% Triton for 60 min. -Incubate the cells with phospho-specific antibody in 50 µl 5% BSA//PBS/0.1% Triton overnight at 4°C. The antibody dilution should be determined experimentally, suggested range 1:100-1:250. -Wash the cells 3x with 200 ul PBS/0.1% Triton for 5 min. -Incubate with secondary antibody HRP-linked, dilution 1:100 in 50^1 5% BSA/PBS/0.1% Triton, for 1 hour at room temperature. -Wash the cells 3x with 200^1 PBS/0.1% Triton for 5 min. -Dissolve 1 tablet of OPD (Sigma) in 25 ml buffer (3.65 g citric acid-H2O and 5.9 g Na2HP04-2H2O in 0.51 H2O, pH 5.6) and add 12.5^1 H2O2. Add 50^1 to each well and incubate for 15 min on a shaker (200 rpm), covered with aluminum foil.

-Stop the reaction with 25^1 H2S04.

-Measure OD490-65o on the ELISA reader.

Mesencephalic dopaminergic neuronal culture Neuronal culture

Neuronal cultures were prepared from the ventral mesencephalon of the fetal rat by enzymatic and mechanical dispersion. The tissue was collected, washed in ice-cold Ca<2+-> and Mg<2+->free phosphate-buffered saline containing 0.6% glucose (PBSG) and incubated for 30 min with PBSG-containing 0.1% trypsin at 37°C . The cell suspension was seeded at a density of 2.5 10<5>cells/cm<2>in 96 well NUNC tissue culture plates. In advance, the culture plates were coated with poly-L-ornithine and CDM-containing 10% fetal calf serum. The cultures were maintained in chemically defined medium (CDM), consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and F12 nutrient supplemented with glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), HEPES (5 mM), insulin (25 µg/ml), human transferrin (100 µg/ml), putrescine (60 µg/ml), sodium selenite (30 nM), streptomycin (100 µg/ml) and penicillin (100 IU/ml ).

Treatment with neurotrophic factors

The neurotrophins were dissolved in 0.5% bovine serum albumin as a stock solution. The neurotrophins were added 3 hours after initial seeding and after 5 days in culture. The same amount of 0.5% bovine serum albumin was added to the control wells.

Uptake of high-affinity dopamine

Dopamine uptake was measured after 10 days. For recording, the cells were washed twice with pre-warmed PBS supplemented with glucose (5 mM), ascorbic acid (100 M) and pargylin (100 M) and pre-incubated for 10 min with the same solution. The pre-incubated solution was replaced with the same solution containing 50 nM [<3>H]DA, and the incubation continued for 15 min at 37°C. Recording was stopped by 3 rapid washes with ice-cold PBS. The accumulated [<3>H]dopamine was released by incubating with acidified ethanol for 30 min at room temperature. The radioactivity was determined after adding 4 ml of scintillation liquid (Packard ultima gold MV) using a Packard scintillation counter. Nonspecific uptake was determined by adding 20 µM cocaine.

The cells were cultured for 10 days in the presence or absence of enovin. Untreated controls were set to 100%. The results are obtained from 1-5 independent experiments.

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List of abbreviations

BLAST search tool for basic local alignment

bp base pair

cDNA complementary DNA

CNS central nervous system

EST expression sequence-tag

EVN enovin

GDNF glial cell line.derived neurotrophic factor GFRa GDNF family receptor a

GPI glycosylphosphatidylinositol

MTC multitissue cDNA

NTN neurturin

PCR polymerase chain reaction

PNS peripheral nervous system

PSP persefine

RT-PCR reverse transcription PCR

TGF-p transforming growth factor p

FISH fluorescent hybridization in situ MTN multiple tissue northern

NGF nerve growth factor

SPR surface plasmon resonance

Claims (27)

1. An isolated nucleic acid molecule encoding a human neurotrophic growth factor designated enovin and having the amino acid sequence illustrated in SEQ ID No.4. 2. A nucleic acid molecule according to claim 1 which is a DNA molecule. 3. A nucleic acid molecule according to claim 1 or 2 which is a cDNA molecule. 4. A nucleic acid molecule according to any one of claims 1 to 3 which has the nucleic acid sequence illustrated in SEQ ID No.2. 5. An isolated human neurotrophic growth factor comprising the amino acid sequence from position 27 to 139 of the amino acid sequence illustrated in SEQ ID No.4. 6. An isolated human neurotrophic growth factor according to claim 5, encoded by a nucleic acid molecule as defined in any of claims 1 to 4. 7. An expression vector comprising a DNA molecule according to any one of claims 2 to 4. 8. An expression vector according to claim 7 comprising a further nucleic acid sequence encoding a reporter molecule. 9. A host cell transformed or transfected with the vector according to any one of claims 7 and 8. 10. A host cell according to claim 9 when the cell is a eukaryotic or bacterial cell. 11. A nucleic acid molecule according to any one of claims 1 to 4 for use as a medicine. 12. Use of a nucleic acid molecule according to any one of claims 1 to 4 in the preparation of a drug to treat or prevent pain syndromes with a mainly peripheral or central neurogenic component, rheumatic/inflammatory diseases and conductance disorders. A neurotrophic growth factor according to any one of claims 5 and 6 for use as a medicine. 14. Use of a neurotrophic growth factor according to any of claims 5 and 6 in the preparation of a drug to treat or prevent pain syndromes with a mainly peripheral or central neurogenic component, rheumatic/inflammatory diseases and conductance disorders. 15. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 1 to 4 together with a pharmaceutically acceptable carrier, diluent or excipient. 16. A pharmaceutical composition comprising a growth factor according to any one of claims 5 and 6, together with a pharmaceutically acceptable carrier, diluent or excipient. 17. An antibody capable of binding to a growth factor according to any one of claims 5 and 6 or an epitope thereof. 18. A pharmaceutical composition comprising an antibody according to claim 17 together with a pharmaceutically acceptable carrier, diluent or excipient. 19. A method for detecting the presence of a growth factor according to any of claims 5 and 6, in a sample, certain method comprising reacting an antibody according to claim 17 with said sample and detecting any binding of said antibody with said growth factor. 20. A method according to claim 19 in which said antibody is conjugated to a reporter molecule. 21. A kit or device for detecting the presence of a neurotrophic growth factor according to any one of claims 5 and 6, in a sample comprising an antibody according to claim 17 and means for reacting said antibody and said sample. 22. A method for identifying an agonist or antagonist of a human neurotrophic growth factor according to any one of claims 5 and 6, said method comprising bringing a cell tissue or an organism expressing a receptor of said growth factor into contact with a candidate compound in the presence of said growth factor and comparing the levels of RET activation in said cell, tissue or organism with a control that has not been in contact with said candidate compound. 23. A method for identifying agonists or antagonists of a neurotrophic growth factor according to any one of claims 5 and 6, said method comprising contacting a cell tissue or an organism expressing an appropriate receptor of said growth factor and cRET with a candidate compound in the presence of said growth factor, measuring the level of activation of a signaling kinase in the signal transduction reaction pathway of which said appropriate receptor is a component, after the addition of an antibody specific for said signaling kinase conjugated to a reporter molecule, compared to a cell tissue or organism that has not been in contact with said connection. 24. A method according to claim 22 or 23 in which said neurotrophic growth factor is enovin, which has the amino acid sequence of SEQ ID No. 3. 25. A method according to any one of claims 22 to 24, wherein said cell tissue or organism is NIH 3T3 cells. 26. A method according to any one of claims 22 to 25 wherein said receptor is any one of GFRa1, GFRa2, GFRa3 or GFRa4. 27. A method according to any one of claims 22 to 26 wherein said antibody is specific for any of p42/p44 MAP kinase, PKB kinase, c-jun, CREB and JNK/SAPIC kinase.